The present invention relates to the field of the detection of Staphylococcus aureus bacteria, and in particular strains of Staphylococcus aureus which are resistant to methicillin.
xe2x80x9cDetectionxe2x80x9d is understood to mean collectively all the techniques which make it possible to identify, qualitatively and/or quantitatively, or to enrich, purify or separate a biological analyte, in this case Staphylococcus aureus bacteria.
In accordance with the document EP-A-0 323 331, a reagent for detecting Staphylococcus aureus bacteria, including the methicillin-resistant strains, is known, this reagent comprising:
fibrinogen,
an antibody recognized by Staphylococcus aureus protein A,
an antibody specifically recognizing the type 5 capsular serotype of Staphylococcus aureus or an antibody specifically recognizing the type 8 serotype of Staphylococcus aureus, and preferably a mixture of these two antibodies.
According to the present invention, a monoclonal or polyclonal antibody is provided which specifically recognizes an epitope common to Staphylococcus aureus strains of various capsular serotypes, particularly the methicillin-resistant strains. This antibody is selected from the type G immunoglobulins, the type M immunoglobulins and the type A immunoglobulins.
A particularly advantageous application of the antibody according to the invention consists in incorporating it into a reagent specific for the detection of Staphylococcus aureus, including the methicillin-resistant strains. Compared with the document EP-A-0 323 331, the sensitivity of the reagent of the invention is superior, because the capacity for recognition of the capsular serotypes is greater.
Thus, according to the invention, there are provided and described:
a monoclonal or polyclonal antibody specific for an epitope common to the Staphylococcus aureus strains of various capsular serotypes, particularly the methicillin-resistant strains, the said antibody being selected from the type G immunoglobulins, the type M immunoglobulins and the type A immunoglobulins; this antibody specifically recognizes at the same time at least two different capsular serotypes of the said Staphylococcus aureus strains; preferably, it is selected from the type G immunoglobulins and the type M immunoglobulins;
a monoclonal or polyclonal antibody as defined above which in particular recognizes at least the type 5 capsular serotype and the type 8 capsular serotype of the said Staphylococcus aureus strains,
a monoclonal antibody capable of being obtained according to a technique adapted from Galfre et al. (Nature, 266, 550-552, (1977)), using as initial cell line for the fusion, the myeloma line SP2/0-Ag14 (ATCC CRL 1581); the fusion is performed with spleen cells from mice of the BALB/C species and of the BALB/CBYJICO strain (marketed by the company IFFA Credo), immunized with a Staphylococcus aureus type 5 strain, according to conventional techniques; the clones obtained were screened by immunoenzymatic techniques (ELISA); the selected clones were reinjected by the intraperitoneal route into mice prepared beforehand for the production of ascitic fluid; each monoclonal antibody was used to sensitize latex particles and was tested for its specificity to recognize Staphylococcus aureus capsular polysaccharides; the monoclonal antibodies called P2G7A1E5 and P6D8D12E1 respectively were selected because of their specificity in recognizing an epitope common to various capsular serotypes, in particular of types 5 and 8;
a monoclonal antibody capable of being obtained, or as obtained from the hybridoma cell line deposited under the No. 96021514, on Feb. 15, 1996 with the ECACC;
a monoclonal antibody capable of being obtained, or as obtained from the hybridoma cell line deposited under the No. 96021513, on Feb. 15, 1996 with the ECACC;
a hybridoma cell line such as deposited under the No. 96021514, on Feb. 15, 1996 with the ECACC, or such as that deposited under the No. 96021513, on Feb. 15, 1996 with the ECACC, or any other derived hybridoma cell line, for example any progeny of this line; the cell lines are claimed as such, as well as any derived cell line, that is to say capable of producing antibodies exhibiting the same immunological characteristics as those described in the present invention;
a specific reagent for the detection of Staphylococcus aureus bacteria and particularly methicillin-resistant strains, comprising at least one antibody specifically recognizing at least one epitope common to the various capsular serotypes of the methicillin-resistant Staphylococcus aureus strains, the said reagent comprising at least one monoclonal or polyclonal antibody as defined above; the said antibody can be attached or coupled, or otherwise, to a support or a marker; monoclonal or polyclonal antibody is understood to mean the antibodies as defined above as well as any antibody exhibiting cross-specificity with the latter;
a specific reagent, particularly for the detection of Staphylococcus aureus bacteria and particularly of methicillin-resistant strains, comprising in addition fibrinogen or a compound based on fibrinogen, capable of being recognized by the Staphylococcus aureus affinity factor for fibrinogen; the fibrinogen or fibrinogen compound can be attached or coupled, or otherwise, to a support or to a marker;
a specific reagent, particularly for the detection of the bacteria Staphylococcus aureus and particularly for the methicillin-resistant strains, comprising in addition immunoglobulins or their Fc fragment recognized by Staphylococcus aureus protein A, attached or coupled, or otherwise, to a support or to a marker.
All sorts of support or marker can be envisaged according to the invention.
Particles in suspension can thus be used. These particles are in particular latex particles such as polystyrene beads or similar particles, preferably having a size of less than 2 xcexcm. By way of example, there may be mentioned Estapor particles, marketed by the company RHONE-POULENC, such as:
polystyrene K080 particles having a diameter of 0.8 xcexcm,
polystyrene K109 particles having a diameter of 0.8 xcexcm,
polystyrene particles having carboxyl groups, PSI 480, having a diameter of 0.8 xcexcm.
Magnetic gels, such as polyacrylamide and/or agarose gels containing magnetic particles can also be used. It is possible, in addition, to use gels such as Ultrogel and Magnogel (trademarks) from the company IBF.
The support used in a reagent according to the present invention may also be red blood cells, for example from sheep.
The support may be in the form of a plate, a cone, a strip, for example made of polystyrene or a styrene-based copolymer, a glass tube or the like.
The attachment of antibodies and of fibrinogen to particles in suspension, particularly of latex, may be achieved according to one of the following techniques:
by passive adsorption, it being possible for the attachment to be spontaneous, during incubation of the latex particles in a solution containing the antibodies and the fibrinogen; an incubation for example of about 2 hours at 20xc2x0 C. is often sufficient;
by covalent bonding, it being possible for the attachment to be achieved by creating a covalent bond between the antibodies and the reactive groups present on some latex particles; it is possible for example to use a carbodiimide to create the covalent bond.
When the support consists of red blood cells, these may be sensitized beforehand with fibrinogen or any other appropriate molecule, according to conventional techniques.
The concentration of antibodies to be attached onto the latex particles, which should be determined for each antibody according to known methods, is usually between 0.1 xcexcg and 100 xcexcg of antibody proteins per millilitre of latex in solution.
In a reagent according to the invention, the antibodies and the fibrinogen may be attached onto a single suspension of particles, for example of latex, or alternatively may be attached onto suspensions of particles which are respectively different, such as red blood cells and latex particles, or various latexes which are then mixed in order to constitute the reagent.
The marker may be any biological or chemical molecule, for example an enzyme, a hapten, an oligonucleotide, a radioactive element and the like.
Other subjects of the invention are the following:
a reagent as defined above comprising an immunoglobulin-type antibody comprising an Fc fragment capable of attaching onto Staphylococcus aureus protein A and/or another compound based on fibrinogen or derivative, capable of reacting with the affinity factor for fibrinogen of the said bacteria, the said reagent further comprising at least one polyclonal or monoclonal antibody capable of specifically recognizing various capsular serotypes of Staphylococcus aureus, particularly types 5 and 8 serotypes;
a process for detecting in a biological sample Staphylococcus aureus bacteria, and particularly methicillin-resistant strains, characterized in that:
a reagent as defined above is made available,
the said sample is brought into contact with the said reagent,
the production of agglutination is observed.